Microlithographic array for macromolecule and cell fractionation

ABSTRACT

A sorting apparatus and method for fractionating and simultaneously viewing individual microstructures, such as free cells, viruses, macromolecules, or minute particles in a fluid medium. The sorting apparatus is composed of a substrate having a receptacle located therein, the receptacle having sidewalls and a floor. An array of obstacles is positioned within the receptacle with the obstacles upstanding from the floor of the receptacle. A transparent cover overlies the array of obstacles to cover the receptacle and afford visual observation of migration of the microstructures exclusively through the array of obstacles. Electrodes may be positioned within the receptacle to generate an electric field in the fluid medium in the receptacle in order to induce the migration of the microstructures. Migration of the microstructures may also occur, for example, by a hydrodynamic field, an optical field, a magnetic field, or a gravity field applied to the receptacle. The obstacles of the array of obstacles may be of various shapes such as round posts, rectangular bunkers, or v-shaped or cup-shaped structures. The arrays of obstacles are formed of a predetermined and reproducible pattern, and can be reused. Methods for manufacturing and using the apparatus are also claimed.

This application is a divisional application of U.S. patent applicationSer. No. 74,432 that was filed on Jun. 8, 1993 now U.S. Pat. No.5,427,663.

BACKGROUND

1. The Field of the Invention

The invention relates to apparatus and methods for fractionatingmicrostructures such as free cells, viruses, macromolecules, or minuteparticles. More particularly, the present invention relates to apparatusand methods for sorting such microstructures in suspension in a fluidmedium while simultaneously viewing individual of those microstructuresduring the process.

2. Background Art

The sizing, separation, and study of microstructures such as free cells,viruses, macromolecules, and minute particles are important tools inmolecular biology. For example, this fractionation process when appliedto DNA molecules is useful in the study of genes and ultimately inplanning and the implementation of genetic engineering processes. Thefractionation of larger microstructures, such as mammalian cells,promises to afford cell biologists new insights into the functioning ofthese basic building blocks of living creatures.

A. Macromolecule Fractionation

While many types of macromolecules may be fractionated by the apparatusof the present invention, the fractionation of a DNA molecule will bediscussed below in detail as one example.

The DNA molecules in a single cell of a complex organism contain all ofthe information required to replicate that cell and the organism ofwhich it is a part. A DNA molecule is a double helical chain of fourdifferent subunits that occur in a genetically coded succession alongthe chain. The four subunits are the nitrogenous bases, adenine,cytosine, guanine, and thymine. The size of such a molecule is measuredby the number of such bases it contains. Natural DNA molecules range insize from a few kilobasepairs in length to hundreds of megabasepairs inlength. The size of a DNA molecule is roughly proportional to the numberof genes the molecule contains.

The size of a DNA molecule can also be expressed by its molecularweight, its length, or the number of basepairs it includes. If thenumber of basepairs is known, that number can be converted into both thelength and the molecular weight of the DNA molecule. One method forestimating the size of small DNA molecules is the process of gelelectrophoresis.

In gel electrophoresis an agarose gel is spread in a thin layer andallowed to harden into a firm composition. The composition comprises afine network of fibers retaining therewithin a liquid medium, such aswater. The fibers of the agarose gel cross and interact with each otherto form a lattice of pores through which molecules smaller than thepores may migrate in the liquid retained in the composition. The size ofthe pores in the lattice is determined generally by the concentration ofthe gel used.

Slots are cast in one end of the gel after the gel is hardened, and DNAmolecules are placed into the slots. A weak electric field of typically1-10 volts per centimeter is then generated in the gel by placing thepositive pole of an electric power source in one end of the gel and thenegative pole of the power source in the opposite end. In DNAelectrophoresis, the negative pole of the power source is placed in thegel at the end of the composition in which the slots containing the DNAare located. The DNA molecules, being negatively charged, are induced bythe electric field to migrate through the gel to the positive pole ofthe power source at the other end of the composition. This occurs atspeeds of typically only a few centimeters per hour.

The electrophoretic mobility of the molecules can be quantified. Theelectrophoretic mobility of a molecule is the ratio of the velocity ofthe molecule to the intensity of the applied electric field. In a freesolution, the mobility of a DNA molecule is independent of the length ofthe molecule or of the size of the applied electric field. In a hinderedenvironment, however, aside from the structure of the hinderedenvironment, the mobility of a molecule becomes a function of the lengthof the molecule and the intensity of the electric field.

The gels used in gel electrophoresis is just such a hinderedenvironment. Molecules are hindered in their migration through theliquid medium in the gel by the lattice structure formed of the fibersin the gel. The molecules nevertheless when induced by the electricfield, move through the gel by migrating through the pores of thelattice structure. Smaller molecules are able to pass through the poresmore easily and thus more quickly than are larger molecules. Thus,smaller molecules advance a greater distance through the gel compositionin a given amount of time than do larger molecules. The smallermolecules thereby become separated from the larger molecules in theprocess. In this manner DNA fractionation occurs.

While gel electrophoresis is a well known and often used process for DNAfractionation, electrophoretic mobility is not well understood in gellattice structures. Thus, the process has several inherent limitations.For example, the pore size in the lattice of gels cannot be accuratelymeasured or depicted. Therefore, the lengths of the molecules migratingthrough the lattice cannot be accurately measured. It has also beenfound that DNA molecules larger than 20 megabasepairs in length cannotbe accurately fractionated in gels. Apparently, the pore size in thelattice of such materials cannot be increased to permit thefractionation of larger molecules, much less even larger particles,viruses, or free cells.

Further, the lattice structure formed when a gel hardens is notpredictable. It is not possible to predict the configuration into whichthe lattice structure will form or how the pores therein will bepositioned, sized, or shaped. The resulting lattice structure isdifferent each time the process is carried out. Therefore, controls andthe critical scientific criteria of repeatability cannot be established.

Gel electrophoresis experiments cannot be exactly duplicated in order topredictably repeat previous data. Even if the exact lattice structuresformed in one experiment were determinable, the structure could stillnot be reproduced. Each experiment is different, and the scientificmethod is seriously slowed.

Also, the lattice structure of a gel is limited to whatever the gel willnaturally produce. The general size of the pores can be dictated to adegree by varying the concentration of the gel, but the positioning ofthe pores and the overall lattice structure cannot be determined ordesigned. Distinctive lattice structures tailored to specific purposescannot be created in a gel.

Further, because the lattice structure arrived at depends upon theconditions under which hardening of the gel occurs, the latticestructure even in a single composition need not be uniform throughout.

Another shortcoming of gel electrophoresis is caused by the fact that agel can only be disposed in a layer that is relatively thick compared tothe pores in its lattice structure, or correspondingly to the size ofthe DNA molecules to be fractionated. Thus, the DNA molecules passthrough a gel in several superimposed and intertwined layers. IndividualDNA molecules cannot be observed separately from the entire group. Eventhe most thinly spread gel is too thick to allow an individual DNAmolecule moving through the gel to be spatially tracked or isolated fromthe group of DNA molecules.

Once a gel has been used in one experiment, the gel is contaminated andcannot be used again. The gel interacts with the materials actually usedin each experiment, and cleaning of the gel for later reuse is notpossible. A gel layer must therefore be disposed after only one use.This also frustrates the scientific objective of repeatability.

Finally, simple gel electrophoresis cannot be used to fractionate DNAmolecules larger than approximately 20 kilobases in length. To overcomethis fact, it is known to pulse the applied electric field to attempt tofractionate longer DNA molecules. This technique, however, results inextremely low mobility and requires days of running time to achievesignificant fractionation. Also, the numerical predictions of thetheories developed to explain the results of this technique dependcritically on the poorly known pore size and distribution in the latticeof the gel.

B. Cell Fractionation

The flexibility of cells is a structural variable of some interest tocell biologists. The flexibility of cells and the effects of variousenvironments on cell flexibility is important to the study of the agingprocess in cells. However, cell fractionation based upon cellflexibility is not easily accomplished in the prior art.

For example, various cells have round or oval shapes with variousdiameters. The shapes are often determined by an underlyingcytoskeleton.

When the cells are circulating in the human body, the cells must, onseveral occasions, pass through variously sized openings andpassageways. This requires substantial flexibility of the cell. Theinability to pass through these openings can be caused by the aging of acell, reactions to specific chemical environments, and other metabolicchanges. When referring to red blood cells, poor red blood cellflexibility results in serious consequences for the larger organism.With respect to cells such as cancer cells, poor flexibility may resultin growth and spread of tumors.

Cancer cells are generally thought to settle in the human body in bloodvessels larger than the cells themselves and stick to those vesselsthrough a special adhesion molecule. As the cancer cells stick to thevessels, new tumors begin to grow. New information, however, hasindicated that the cancer cells move too quickly to become adhered tothe vessels in this fashion. It is now thought that cells may start newtumors when they become stuck in vessels too narrow for the cancer cellsto pass through. The flexibility of the cancer cells is important indetermining the deleterious effect of the cell.

Three physical limitations impinge on the flexibility of many cells.First, many cells must maintain both a constant volume V and a constantsurface area A as it deforms. Second, the cell membrane, while veryflexible, cannot increase in area. It will tear, if forced to do so.Third, as a cell ages it loses membrane and the surface-to-volume ratiodecreases.

For example, a biconcave red blood cell has a maximum diameter of about8 microns, a surface area of about 140 microns square, and a volume ofabout 95 microns cube in the normal state. It can be shown that formature red blood cells for openings smaller in diameter thanapproximately 3 microns, the constraints of constant volume V andsurface area A cannot be met. The passage of a red blood cell through apassageway of that size, thus, cannot occur without membrane rupture.Since the smallest capillary openings are but approximately 3.5 microns,red blood cells passing through the capillary bed are uncomfortablyclose to being ruptured. Accordingly, small changes in the physicalvariables that control deformability can lead to microangiopathy andsevere organism distress.

There exist several techniques for measuring cell flexibility anddeformability. These range from the elegant and pioneering micropipetteaspiration techniques, to the nucleopore filtration and laminar stresselongation techniques. The latter are termed ektacytometry. All are veryuseful and have provided an excellent initial database for studying redblood cell deformation, but each has certain weaknesses.

The micropipette aspiration technique can only study one cell at a time.The nucleopore filtration technique does not allow observation of cellsduring the actual passage thereof through openings. Ektacytometry doesnot deform cells in narrow passages.

OBJECTS AND SUMMARY OF THE INVENTION

It is accordingly a broad objective of the present invention to providean improved method and apparatus for fractionating microstructures, suchas macromolecules, viruses, free cells, and minute particles.

Another object of the present invention is to facilitate research intothe behavior and structure of macromolecules, such as DNA molecules,proteins and polymers.

Correspondingly it is an object of the present invention to enhance theeffectiveness of electrophoresis techniques currently applied to thefractionation of such macromolecules.

Yet another object of the present invention is to permit fractionationof DNA molecules in excess of 20 megabasepairs in length, withoutresorting to the use of a pulsed electric field.

Yet another object of the present invention is to provide a hinderedenvironment in which to conduct macromolecular electrophoresis, whereinthe lattice structure of the hindered environment can be designed atwill and replicated with repeatable consistency.

Another object of the present invention is to provide such a latticestructure in which the distribution, size, and shape of the pore thereinare substantially uniform.

Yet another object of the present invention is to provide an apparatusfor fractionating macromolecules while simultaneously observingindividual of the macromolecules during the process.

Yet another object of the present invention is to advance the study ofthe structure and mechanics of free cells, such as red blood cells,cancer cells, and E. Coli cells.

It is yet another object of the present invention to provide anapparatus for fractionating cells according to the elasticity thereofand other physical properties which are otherwise difficult to probe bybiological markers.

In particular, it is an object of the present invention to provide amethod and apparatus for observing cell behavior during the passage ofcells through channels in essentially a single layer in single file.

Yet another object of the present invention is to provide an apparatusfor sorting and viewing microstructures, which is not contaminated bythe microstructures being sorted.

Yet another object of the present invention is to increase the mobilityof large molecules during electrophoresis.

Additional objects and advantages of the invention will be set forth inthe description which follows, and in part will be obvious from thedescription, or may be learned by the practice of the invention. Theobjects and advantages of the invention may be realized and obtained bymeans of the instruments and combinations particularly pointed out inthe appended claims.

To achieve the foregoing objects, and in accordance with the inventionas embodied and broadly described herein, a sorting apparatus isprovided for fractionating and simultaneously viewing individualmicrostructures such as free cells, viruses, macromolecules, or minuteparticles in a fluid medium. The sorting apparatus allows themicrostructures to be observed in essentially a single layer and wherebya particular microstructure can be tracked throughout. One embodiment ofan apparatus incorporating the teachings of the present inventioncomprises a substrate having a shallow receptacle located on a sidethereof. The receptacle has first and second ends and a floor bounded onopposite sides by a pair of upstanding opposed side walls extendingbetween the first and second ends of the receptacle. Migration of themicrostructures from the first end of the receptacle to the second endof the receptacle defines a migration direction for the receptacle. Theheight of the side walls defines a depth of the receptacle. The depth iscommensurate with the size of the microstructures in the fluid medium,whereby the microstructures will migrate in the fluid through thereceptacle in essentially a single layer.

According to one aspect of the present invention, the array furthercomprises sifting means positioned within the receptacle intermediatethe first and second ends traversing the migration direction. Thesifting means are for interacting with the microstructures to partiallyhinder migration of the microstructures in the migration direction inthe fluid medium.

In one embodiment of such a sifting or hindered environment means, anarray of obstacles is provided upstanding from the floor of thereceptacle. The array of obstacles is arranged in a predetermined andreproducible pattern. The obstacles may comprise posts, bunkers,v-shaped and cup-shaped structures, and other shapes of structures. In apreferred embodiment, the receptacle and array of obstacles therein aresimultaneously formed on a side of the substrate using microlithographytechniques.

According to another aspect of the present invention, the apparatusfurther comprises ceiling means positioned over the sifting means forcovering the receptacle and for causing migration of the microstructuresin essentially a single, layer through the sifting means exclusively.The ceiling means are so secured to the sifting means as to precludemigration of microstructures between the sifting means and the ceilingmeans. In one embodiment of an apparatus incorporating the teachings ofthe present invention, such a ceiling means comprises a coverslip whichextends across the substrate from one of the pair of upstanding opposingside walls to the other of the pair of upstanding opposed side wallswith the tops of the obstacles in the array bonded to the adjacent sideof the coverslip. Optimally, the coverslip and the substrate havesimilar thermal coefficients of expansion. Also, preferably thesubstrate and the array of obstacles are comprised of a material that isnoninteractive in a normal range of temperatures with themicrostructures to be fractionated therein.

Optionally, the coverslip may be transparent, thereby to afford forvisual observation of the microstructures during sorting. Thetransparent form of the coverslip represents one example of a structurecapable of performing the function of what will hereinafter be referredto as a "capping means" for the present invention.

In another aspect of an apparatus incorporating the teachings of thepresent invention, the array includes electric force means forgenerating in the receptacle an electric field used to induce chargedmicrostructures to migrate through the fluid medium from one end of thereceptacle to the other. In one embodiment, such an electric force meansmay comprise a first electrode positioned at the first end of thereceptacle and a second electrode positioned at the second end of thereceptacle. The electrodes may comprise metal strips disposed on thefloor of the receptacle. A power source is electrically coupled betweenthe first and second electrodes.

In yet another aspect of the present invention, an apparatusincorporating the teachings thereof further comprises sensor meanspositioned within the array of obstacles for sensing the intensity ofthe electric field generated within the array. The sensor means mayoptionally be electrically coupled with the electric force means to varythe intensity of the electric field in a predetermined manner. In oneembodiment of the sensor means, first and second sensor electrodes arepositioned within the array of obstacles, and control means are coupledto the first and second electrodes for maintaining the electric field inthe array at a predetermined intensity.

In one embodiment of the present invention, such a control meansincludes a differential amplifier circuit having first and second inputterminals coupled respectively to the first and second sensorelectrodes. The differential amplifier circuit produces an output signalcorresponding to the intensity of the electric field in the arraybetween the first and second sensor electrodes. Comparator means arecoupled to the differential amplifier for producing a control signalreflecting the difference between the output signal of the differentialamplifier and a reference voltage reflecting the predetermined intensityof the electric field in the array. Driver means are coupled to thecomparator means for varying the intensity of the electric field inaccordance with the control signal produced by the comparator means.

The present invention also contemplates a method for manufacturing anapparatus as described above. In the method a receptacle is formed onone side of a substrate having a floor bound by a pair of upstandingopposing side walls. An array of obstacles are built within thereceptacle. Preferably the step of forming the receptacle and the stepof building the array are performed simultaneously. To do so, aphotoresist layer is positioned over areas of the substrate intended tocorrespond to the tops of the obstacles of the arrays. Then thesubstrate is etched to a predetermined depth equal to the depth of thereceptacle. The receptacle with the array of obstacles upstandingtherein is formed as a result. The photoresist layer is then dissolvedfrom the substrate.

Ultimately the method of the present invention includes the step ofsecuring a transparent coverslip to the top of each of the obstacles. Todo so the coverslip is positioned over the array of obstacles in contactwith the top of each. An electric field is applied between the coverslipand the array of obstacles.

The present invention also contemplates a method for sorting andsimultaneously viewing individual microstructures. In that method themicrostructures are placed in a fluid medium and introduced into one endof an apparatus as described above. The microstructures are then inducedto migrate in the fluid through the array of obstacles and visuallyobserved during the process.

An additional embodiment within the scope of the present inventioncomprises an apparatus for sorting and simultaneously viewing cells in afluid medium in order to study flexibility of cells and the effects ofvarious environment on cells. The apparatus comprises a substrate havinga shallow receptacle located on a side thereof and channeling meanspositioned within the receptacle for allowing passage of cells throughthe receptacle in essentially a single layer and in single file. In oneembodiment of the present invention, such a channeling means comprisespassageways positioned within the receptacle through which the cells maypass.

The apparatus can be used to measure the amount of energy consumedduring movement of the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the manner in which the above-recited and otheradvantages and objects of the invention are obtained, a more particulardescription of the invention briefly described above will be rendered byreference to a specific embodiment thereof which is illustrated in theappended drawings. Understanding that these drawings depict only atypical embodiment of the invention and are not therefore to beconsidered limiting of its scope, the invention will be described andexplained with additional specificity and detail through the use of theaccompanying drawings in which:

FIG. 1 is a perspective view of one embodiment of a sorting apparatusincorporating the teachings of the present invention;

FIG. 2 is an exploded view of the apparatus illustrated in FIG. 1 withthe transparent coverslip thereof shown separated from the substrate tomore fully reveal an array of obstacles therebetween;

FIG. 3 is an enlarged view of the obstacles within the area of the arrayof FIG. 2 encircled by line 3--3 therein;

1 FIG. 4 is a further enlarged view of the obstacles within the area ofthe array of FIG. 3 encircled by line 4--4 therein;

FIG. 4A is an elevational cross section view of two of the obstaclesillustrated in FIG. 4 and the lattice pore therebetween taken alongsection line 4A--4A shown in FIG. 4;

FIGS. 5A-5F illustrate the steps in a method for manufacturing a sortingapparatus, such as the sorting apparatus illustrated in FIG. 1;

FIG. 6 is a plan view of the sorting apparatus shown in FIG. 1 with theobstacles enlarged to illustrate DNA molecules migrating through thearray;

FIG. 7 is a plan view of an alternate embodiment of obstacles usable inan array in a sorting apparatus incorporating the teachings of thepresent invention wherein the obstacles are v-shaped;

FIG. 8 is a plan view of another alternate embodiment of obstaclesusable in an array in a sorting apparatus incorporating the teachings ofthe present invention wherein the obstacles are cup-shaped;

FIG. 9 is a plan view of yet another embodiment of a sorting apparatusincorporating the teachings of the present invention in which a pair ofsensor electrodes are located within the array of the sorting apparatus;

FIG. 10 is a cross sectional elevation view of the apparatus shown inFIG. 9 taken along section line 10--10 shown therein, illustratingpositioning of the top sensor electrode within the array of obstacles;

FIG. 11 is a cross sectional elevation view of the apparatus shown inFIG. 9 taken along section line 11--11 shown therein, illustratingpositioning of the top sensor electrode;

FIG. 12 is a cross sectional elevation view of the sensing apparatusshown in FIG. 9 taken along section line 12--12 shown therein,illustrating positioning of the top sensor electrode outside of thearray of obstacles;

FIG. 13 is an electrical schematic diagram of the feedback circuitassociated with the pair of sensor electrodes shown in the embodiment ofthe sensing apparatus illustrated in FIG. 9;

FIG. 14 is an enlarged plan view of another embodiment within the scopeof the present invention illustrating a portion of a percolating arrayhaving cells migrating therein;

FIG. 15 is a perspective enlarged view of an alternate embodiment ofobstacles for an array in a sorting apparatus incorporating theteachings of the present invention to stimulate cell behavior simulatingthe passage of such cells through the passageways in the human body;

FIG. 16 is a plan view of an embodiment of a sorting apparatusincorporating the teachings of the present invention utilizing obstaclesof the type shown in FIG. 15 enlarged to illustrate cells deforming tomigrate through the array thereof;

FIGS. 17A-17E illustrate in detail the movement of a healthy round cellbetween two adjacent obstacles of the type illustrated in FIGS. 15 and16; and

FIGS. 18A-18B illustrate in detail the movement of an unhealthy cellunable to deform and pass through the restriction formed by two adjacentobstacles of the type illustrated in FIGS. 15 and 16.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a method and apparatus that facilitatesthe fractionation of many types of microstructures. For example, thepresent invention allows successful fractionation of extremely long DNAmolecules of chromosomal length in low quantities, such as even singlemolecules. The present invention also facilitates the fractionation ofmuch larger microstructures, such as red blood cells.

Each application will be described in turn below.

A. Macromolecule Fractionation

Although reference will be made herein to the fractionation of DNAmolecules, it should be noted that fractionation of other macromoleculesand microstructures, such as proteins, polymers, viruses, cells, andminute particles, is considered to be within the scope of the presentinvention.

The diffusion of long polymers in complex environments where themobility of the polymer is greatly perturbed is both a challengingstatistical physics problem and a problem of great importance in thebiological sciences. The length fractionation of charged polymers, suchas DNA in gels, is a primary tool of molecular biology. One of the mainstumbling blocks to understanding quantitatively the physical principlesbehind the length-dependent mobility of long polymers in complexenvironments has, however, been the ill-characterized nature of thehindering environment, the gel. It is possible, however, using thepresent invention to generate complex environments which are very wellcharacterized and consistently reproducible.

Referring to FIG. 1, a sorting apparatus 20 is illustrated forfractionating and simultaneously viewing microstructures such as freecells, macromolecules, and minute particles in a fluid medium inessentially a single layer. Sorting apparatus 20 is comprised of asubstrate 22 having a shallow receptacle 24 located on a side 26thereof. In the embodiment shown, receptacle 24 is recessed in side 26of substrate 22, although other structures for producing a recess suchas receptacle 24 would be workable in the context of the presentinvention.

Receptacle 24 includes a floor 28 shown to better advantage in FIG. 2bounded by a pair of upstanding opposing side walls 30, 31 and a firstend 32 and a second end 34. The height of side walls 30, 31 define adepth of receptacle 24. The depth of receptacle 24 is commensurate withthe size of the microstructures to be sorted in sorting apparatus 20.The depth of receptacle 24 is specifically tailored to cause thosemicrostructures in a fluid medium in receptacle 24 to form essentially asingle layer. Thus, when the microstructures are caused to migrate inthe fluid medium through receptacle 24, the microstructures do so inessentially the single layer. The migration of the microstructuresoccurs in a migration direction indicated by arrow M defined relative tosorting apparatus 20.

Substrate 22 may be comprised of any type material which can bephotolithographically processed. Silicon is preferred; however othermaterials, such as quartz and sapphire, can also be used.

In accordance with one aspect of the present invention, ceiling meansare provided for covering receptacle 24 intermediate first end 32 andsecond end 34 thereof and for causing the migration of themicrostructures within receptacle 24 to occur in essentially a singlelayer. As shown by way of example and not limitation, in FIG. 1, acoverslip 36 extends across receptacle 24 in substrate 22 from one ofthe pair of upstanding opposing side walls 30 to the other of said pairof upstanding opposing side walls 31. The manner by which coverslip 36is bonded to side 26 of substrate 22 will be discussed in detailsubsequently.

According to one aspect of the present invention, a sorting apparatus,such as sorting apparatus 20, is provided with sifting or hinderedenvironment means positioned within receptacle 24 traversing themigration direction associated therewith for interacting with themicrostructures to partially hinder the migration of the microstructuresin the migration direction.

As is suggested in the exploded view of FIG. 2, one form of such asifting or hindered environment means utilizable in accordance with thepresent invention is an array 38 of minute obstacles 39 upstanding fromfloor 28 of receptacle 24. Obstacles 39 are sized and separated as toadvance the particular sorting objective of sorting apparatus 20. Themanner of forming obstacles 39 of array 38, as well as a number ofexamples of embodiments of obstacles utilizable in such an array, willbe discussed in substantial detail below.

Coverslip 36 is so secured to the top of obstacles 39 in array 38 as topreclude migration of microstructures between the obstacles 39 andcoverslip 36. Coverslip 36 may optionally be transparent. In this form,coverslip 36 performs not only the function of the ceiling meansdescribed above, but also performs the function of a capping means forcovering a shallow receptacle, such as receptacle 24, and for affordingvisual observation therethrough of the migration of microstructuresthrough array 38. Coverslip 36 may be comprised of any ceramic material.Pyrex is preferred, but other materials such as quartz and sapphire, forexample, may also be used.

In accordance with another aspect of the present invention, a sortingapparatus, such as sorting apparatus 20, is provided with electric forcemeans for generating an electric field in the fluid medium in receptacle24. The electric field induces the microstructures to migrate throughthe fluid medium, either from first end 32 to second end 34 or fromsecond end 34 to first end 32, depending upon the polarity of theelectric field and whether the microstructures are positively ornegatively charged. Negatively charged microstructures, such as DNAmolecules, will be induced to flow toward the positive pole. Positivelycharged microstructures, such as proteins, will be induced to flowtoward the negative pole.

By way of example and not limitation, a first electrode 40 is shown inFIG. 2 as being located in first end 32 of receptacle 24 and a secondelectrode 42 located in second end 34 of receptacle 24. First electrode40 and second electrode 42 each comprise a metal strip disposed on floor28 of receptacle 24. In the preferred embodiment, the metal strip isformed from evaporated gold.

A battery 44, or other power source is electrically coupled betweenfirst and second electrodes, 40 and 42, such that first electrode 40comprises a negative pole and second electrode 42 comprises a positivepole. The electric field generated between first and second electrodes,40 and 42, is non-alternating, but the use of an alternating powersource in place of battery 44 would be consistent with the teachings ofthe present invention.

When DNA is the microstructure being induced to migrate, the electricfield intensity in receptacle 24 is in the range of from about 0.1 voltper centimeter to about 10 volts per centimeter. In the preferredembodiment, an electric field intensity is about 1.0 volt percentimeter.

Referring now to FIG. 3, the portion of FIG. 2 encircled by line 3--3 isseen illustrated in an enlarged manner. FIG. 3 illustrates one exampleof a sifting or hindered environment means for use in a sortingapparatus of the present invention. As shown, array 38 comprises aplurality of obstacles 39 upstanding from floor 28 of receptacle 24.Although FIG. 3 illustrates obstacles 39 as being positioned withinarray 38 in an ordered and uniform pattern, it is within the scope ofthe present invention to have a staggered pattern, or any desiredpredetermined and reproducible pattern.

FIG. 4 illustrates the various dimensions of a typical obstacle 39. Theheight H of obstacle 39 is measured in a direction normal to floor 28 ofreceptacle 24. The length L of obstacle 39 is measured in a directionparallel to said migration direction M. The width W of obstacle 39 ismeasured in a direction normal to the migration direction M. Each of theobstacles 39 are separated from an adjacent obstacle 39 by apredetermined separation distance S_(d). The space between adjacent ofobstacles 39 in a cross section of array 38 taken normal to floor 28 ofreceptacle 24 defines a pore 54 of the lattice structure cumulativelyproduced by obstacles 39 of array 38. For convenience of reference inFIG. 4, such a typical pore 54 has been shaded, but will be discussed inadditional detail subsequently. These dimensions can be changed anddesigned to be as desired depending upon the type and size ofmicrostructure to be sorted, the design of the array, and the type ofobstacles in the array.

For example, the separation distance S_(d) will vary depending uponwhether the migration of microstructures through pores 54 are DNAmolecules, viruses and bacterial cells, or mammalian cells. Formigration of DNA molecules, the separation distance S_(d) is within therange of about 0.01 microns to about 20.0 microns. For migration ofviruses and bacterial cells, the separation distance S_(d) is within therange of about 0.01 microns to about 1.0 micron. For migration ofmammalian cells, the separation distance is within the range of fromabout 1.0 micron to about 50.0 microns. It is presently preferred thatthe separation distance S_(d) be substantially equal to the radius ofgyration of the molecule, the radius of gyration being the distancewalking out from the center of the molecule.

Length L also varies depending upon the microstructure to be migratedthrough array 38 of obstacles 39. In a presently preferred embodiment,the length is generally equal to the separation distance. With regard toheight H, the height of obstacles may generally be in the range of from0.01 microns to about 20.0 microns. For smaller microstructures, theobstacles may have a height in a range from about 0.01 microns to about0.50 microstructures. For larger microns, the height may be in the rangefrom about 1.0 micron to about 5.0 microns.

FIG. 4A, a cross-section of two obstacles 39, illustrates in planar viewa typical pore 54. Pore 54 comprises the area defined by two obstacles39 through which a microstructure must pass. Pore 54 is defined by theheight H and the separation distance S_(d) between the obstacles. Thedesired size of pore 54 is determined by reference to the size of themicrostructures to be sorted therethrough. An important aspect of theapparatus of the present invention is that not only is the pore size ofthe arrays known, but it is also constant and reproducible. More stabledata can be obtained.

The characteristic number which sets the length scale for theconformation of a polymer in solution is the persistence length given bythe equation: ##EQU1## where E is the Young's modulus,

I_(A) is the surface moment of inertia,

K_(B) is Boltmann's constant, and

T is the absolute temperature.

For DNA at normal physiological salt concentrations and pH, about 0.1MNaCl and pH 7.6, P is 0.06 microns. If the etch depth of the array isapproximately equal to or less than P then the polymer can be viewed asmoving in a quasi-two-dimensional environment, as is the case in theapparatus used within the scope of the present invention.

In one preferred embodiment of a sorting apparatus, such as sortingapparatus 20, incorporating the teachings of the present invention,substrate 22 is provided with a receptacle 24 having sides 30 and 31 ofapproximately 3.0 millimeters in length and first and second ends 32,34, respectively, of approximately 3.0 millimeters in length. Each ofobstacles 39 has a height H of approximately 0.1 microns, a width W ofapproximately 1.0 micron, a length L of approximately 1.0 micron and aseparation distance S_(d) of approximately 2.0 microns. These sizes willvary depending upon the microstructure to be sorted, bearing in mindthat obstacles 39 should be so sized and separated in array 38 thatmicrostructures migrate through array 38 of obstacles 39 in essentiallya single layer.

The method of making the apparatus of the present invention involvesforming receptacle 24 on one side of substrate 22. Receptacle 24 shouldbe formed of a size such that microstructures migrate in the fluidthrough receptacle 24 in essentially a single layer. A further stepcomprises creating array 38 of obstacles 39 within receptacle 24. Eachof obstacles 39 have a top 56, sides 57, and a bottom end 58. Obstacles39 are upstanding from floor 28 of receptacle 24 in a predetermined andreproducible pattern. In one preferred embodiment, the array ofobstacles comprises a plurality of posts.

By way of example and not limitation, the creation of posts within thereceptacle is illustrated in FIGS. 5A-5F. As shown in FIG. 5A, theforming step comprises developing a photosensitive photoresist layer 60over areas of substrate 22 that are intended to correspond to tops 56 ofobstacles 39. This is accomplished by exposing substrate 22 to lightthrough a mask having thereon a corresponding opaque pattern.

The portion of photoresist layer 60 which is exposed to light becomessoluble in a basic developing solution, while the unexposed portionremains on substrate 22 to protect substrate 22. Thus, after developmentin the developing solution, substrate 22 is left with a pattern ofphotoresist layer 60 that is identical to the opaque pattern of themask. FIG. 5B illustrates substrate 22 with photoresist layer 60 thereonafter exposure to light and development in solution.

The next step comprises etching substrate 22 such that the areas ofsubstrate 22 unshielded by photoresist layer 60 are exposed to theetching, thereby forming receptacle 24. The array 38 of obstacles 39upstanding within the etched receptacle 24 is formed by the portions ofsubstrate 22 shielded by photoresist layer 60. FIG. 5C illustratesformation of receptacle 24 and the obstacles 39.

As can be seen in FIG. 5C, as the substrate 22 is etched, thephotoresist layer 60 is also etched, but at a slower rate. FIG. 5Cillustrates the receptacle 24 half formed, and photoresist layer 60partially etched away. If, for example, the photoresist layer is etchedat a rate 1/10 the rate that substrate 22 is etched, the resultingreceptacle can at most have a depth 10 times the thickness of thephotoresist layer. The thickness of photoresist layer 60 must thereforebe chosen accordingly.

The etching process can be terminated at any time when the desired depthof the receptacle is reached. As illustrated in FIG. 5D, there may besome photoresist layer 60 still present on substrate 22 when the etchingis terminated. If so, the next step is then dissolving photoresist layer60 from substrate 22. This step leaves a clean substrate 22 as shown inFIG. 5E.

Within the scope of the present invention, etching may occur by manytypes of methods. In the preferred embodiment, ion milling is used suchthat an overhead ion beam is used to etch the substrate 22 andphotoresist layer 60. Other methods of etching, such as chemicaletching, are also within the scope of the present invention.

Turning now to FIG. 5F, the step of fusing coverslip 36 to substrate 22is illustrated. In the preferred embodiment within the scope of thepresent invention, the step comprises positioning coverslip 36 overarray 38 of obstacles 39 such that coverslip 36 is in contact with eachof obstacles 39, and then applying an electric field between coverslip36 and each of obstacles 39. The coverslip 36 is held with a negativepotential. The obstacles 39 are held at a positive potential. Ions arethereby induced to migrate there between to create a bond betweencoverslip 36 and each of obstacles 39 at all areas of contact. Theprocess of this step is referred to as field assisted fusion.

The voltage used to fuse coverslip 36 to the substrate 22 is preferablyabout 1 kilovolt but can be within the range of from 200 volts to about2000 volts. The time for fusion is about 30 minutes at a temperature ofabout 400° C. The temperature can also range from about 300° C. to about600° C., with 400° C. being the preferred temperature. In the preferredembodiment within the scope of the present invention, the coverslipcomprises a pyrex material. However, any transparent ceramic may beused. For example, sapphire and quartz are material which may also beused for the coverslip.

It is preferred that the material used for coverslip 36 havesubstantially the same coefficient of thermal expansion as substrate 22.Otherwise, at the high temperature of fusion, the coverslip 36 and thesubstrate 22 will expand at different rates and a seal between the twowould be difficult or impossible to accomplish.

Successful fusion can be tested by injecting a fluorescent fluid intothe apparatus. A completely fused coverslip will not allow passage ofany fluorescent fluid between coverslip 36 and obstacles 39.

FIG. 6 illustrates one use of an embodiment of the present invention. Asearlier stated, the apparatus of the present invention can be used forcharged macromolecular electrophoresis. For example, the apparatus maybe used to conduct protein electrophoresis, and DNA electrophoresis,with the positive and negative poles adjusted accordingly. FIG. 6illustrates DNA electrophoresis.

As illustrated in FIG. 6 by way of example and not limitation, DNAmolecules 68 are placed into a buffer solution and placed into a loadingarea 66 positioned on the first end 32 of receptacle 24. Loading area 66comprises a portion of receptacle 24 where no obstacles 39 have beenformed. Buffer is also added to a second loading area 67 positioned onsecond end 34. Second loading area 67 also comprises a portion ofreceptacle 24 where no obstacles have been formed. The loading areas arethen covered.

Once DNA molecules 68 have been positioned, battery 44 is engaged and anelectric field is generated. The electric field is so polarized as toinduce the negatively charged DNA microstructures to migrate through thefield from first electrode 40 toward second electrode 42 in receptacle24.

As DNA molecules 68 migrate from first end 32 toward the second end 34,their movements are hindered by the array 38 of obstacles 39 upstandingwithin receptacle 24. Interaction between obstacles 39 and DNA molecules68 are illustrated in FIG. 6.

In FIG. 6, DNA molecules 68 are illustrated as long arrows. Thedirection of the arrows indicates the direction of migration of DNAmolecules 68. As DNA molecules migrate through array 38 of obstacles 39,large bodies of DNA molecules may become hooked by obstacles 39 and maybecome trapped. The hooked and trapped DNA molecules are labelled as68a. When, as illustrated in FIG. 6, obstacles 39 are posts, DNAmolecules 68 stretch around obstacles 39 as they become hooked. Theobstacles are thought to catch the large DNA molecules and hold themagainst the electric field. Some DNA molecules 68 may stretch andrelease themselves from the obstacles. Smaller DNA molecules possesssufficient Brownian motion to release themselves.

It is an important feature of the present invention that any pattern ofarray 38 of obstacles 39 can be designed within the scope of the presentinvention. The array 38 can comprise an ordered, evenly spaced formationwherein the obstacles are positioned in uniform rows and columns.Alternatively, array 38 may comprise a staggered formation whereinpositioning of the obstacles is not uniform but rather scattered aroundthe array. Further, array 38 may comprise a mixture of such arrangementsdisposed along migration direction M traversing same.

The design of the array can be formulated to correspond to any specificintended use. The ordered, evenly spaced configuration can be used forimaging of long megabase DNA fragments. The staggered configuration,having a higher possibility of hooking the DNA molecules as the DNAmolecules migrate through the array, can be used to more directly testthe role of DNA relaxation and hooking in the mobility of DNA molecules.

The shapes of the obstacles may also vary within the scope of thepresent invention. Illustrated in FIG. 7 is an array 70 of v-shapedobstacles 72 upstanding from floor 28 of receptacle 24, and having av-shaped cross section in a plane disposed parallel to floor 28 ofreceptacle 24. Arms 73 and 74 intersect at one end to form a vertex 75and an open end 76. The open end 76 of said v-shaped cross section ofv-shaped obstacles 72 is disposed opposing migration direction M ofreceptacle 24.

The size of v-shaped obstacles 72 should be such that as microstructuresof various sizes migrate through the array 70 of v-shaped obstacles 72in a direction M, the microstructures are hindered and trapped withinthe open end 76 of v-shaped obstacles 72. Smaller v-shaped obstacles 72will trap small microstructures while larger v-shaped obstacles 72 willtrap both the smaller and the larger microstructures.

It is conceivable that various sizes of v-shaped obstacles 72 may beused within one array 70. For example, smaller v-shaped obstacles 72 maybe positioned toward the first end 32 of receptacle 24 with largerv-shaped obstacles 72 positioned toward the second end 34 of receptacle24. Thus, as the microstructures migrate from first end 32 toward secondend 34, the smaller microstructures will become trapped in the smallerv-shaped obstacles 72 while the larger microstructures will flow pastthe smaller v-shaped obstacles 72. As the larger microstructures flowthrough the larger sized v-shaped obstacles 72, the largermicrostructures will also become trapped. The microstructures will thenbe separated with respect to size.

Referring now to FIG. 8, an alternate embodiment of the array ofobstacles within the scope of the present invention is illustrated. FIG.8 illustrates an array 78 of obstacles 80 which are cup-shaped.Obstacles 80 have a cup-shaped cross-section in a plane disposedparallel to floor 28 of receptacle 24.

As illustrated, cup-shaped obstacles 80 may comprise a first leg 82 anda second leg 84 substantially parallel to the direction of migration ofthe microstructures, and a third leg 86 substantially perpendicular tothe direction of migration. First, second, and third legs, 82, 84, and86, respectively, are positioned such that they define an open end 88into which the microstructures can become trapped as the microstructuresmigrate through the cup-shaped obstacles 80. As with v-shaped obstacles72, various sizes of cup-shaped obstacles 80 may be positioned withinarray 78 in any pattern desired. The open end 88 of the cup-shapedcross-section is disposed opposing migration direction M of receptacle24.

It is important to note that whatever type of array is used, the arrayis reproducible. Additionally, an optimum design can be perfected overtime by making minor changes to the arrays for each new experiment untilthe most preferred design is obtained.

Referring now to FIG. 9, and in accordance with another aspect of thepresent invention, a sorting apparatus 110 is comprised of an apparatus,such as sorting apparatus 20, further provided with sensor means fordetecting the intensity of the electric field generated within the arrayof obstacles, such as array 38 of obstacles 39, between any determinedfirst and second points therein, to enable control of the intensity ofthe electric field.

Sorting apparatus 110 is illustrated in FIG. 9. As in sorting apparatus20, shown in FIGS. 1 and 2, sorting apparatus 110 includes firstelectrode 40 and second electrode 42, functioning as negative andpositive poles, for an electric field generated therebetween. That fieldmay be non-alternating, by coupling therebetween a battery, such asbattery 44 of FIGS. 1 and 2. Nevertheless, it would also be consistentwith the teachings of the present invention to develop an electric fieldthat is alternating or switchable as to polarity, either selectively oraccording to some repeated pattern. In the case of sorting apparatus110, however, the electric field developed between first and secondelectrodes 40 and 42 is produced by a feedback varied drive voltagecircuit 144 that will be explored in detail subsequently.

First electrode 40 comprises a metal strip positioned along floor 28 ofreceptacle 24 at first end 32. First electrode 40 is soldered tosubstrate 22 and to various lead lines at a first area 128. Secondelectrode 42 comprises a metal strip positioned along floor 28 ofreceptacle 24 at second end 34. Second electrode 42 is soldered tosubstrate 22 and to various lead lines at a second area 129. In thepreferred embodiment, the metal strips, first and second electrodes 40and 42, comprise gold evaporated into floor 28.

Positioned within the array is sensor means for detecting the intensityof the electric field generated between first electrode 40 and secondelectrode 42 between predetermined first and second points therein. Thesensor means enables control of the intensity of the electric fieldgenerated.

The sensor means comprises a first sensor electrode 130 positionedwithin array 38 of obstacles 39 at the first predetermined point 134.The sensor means further comprises a second sensor electrode 132 whichis positioned within array 38 of obstacles 39 at the secondpredetermined point 135. First sensor electrode 130 is positioned withinarray 38 toward first end 32 of receptacle 24 in a first sensor channel138 formed along floor 28 of receptacle 24. No obstacles 39 are presentwithin channel 138. A clear area is formed wherein the sensor electrodeis positioned.

In one embodiment of the present invention, the array 38 is turned at a45 degree angle before the sensor electrodes are positioned within thearray.

As can be seen in FIG. 9, first and second sensor electrodes, 130 and132, extend through sidewall 31 of receptacle 24, past coverslip 36, andonto substrate 22. Positioning of first sensor electrode 130 can be seenin FIGS. 10-12.

In FIG. 10, first sensor electrode 130 is shown disposed along floor 28of receptacle 24 within first sensor channel 138. Obstacles 39 can beseen positioned along the sides of top sensor channel 138, but notwithin channel 138 itself. Coverslip 36 is shown fused to the obstacles39 and covering channel 138.

FIG. 11 illustrates channel 137 extending away from sidewall 31 ofreceptacle 24. Obstacles are not present within channel 137. Coverslip36 is illustrated in FIG. 11 as positioned over channel 137.

FIG. 12 illustrates the first sensor soldering area 140 where firstsensor electrode 130 is soldered to the substrate 22 and connected tofirst sensor lead 152, to be later discussed in more detail.

Although cross sections for only first sensor electrode 130 are shown,it must be noted that second sensor electrode 132 is positioned withinapparatus 110 in the same fashion. Second sensor electrode 132 ispositioned within a bottom sensor channel 139 within the array 38 ofobstacles 39. Second sensor electrode 132 is soldered to substrate 22and connected to a second sensor lead 154 at a second sensor solderingarea 142. Second sensor lead 154 will be later discussed in more detail.

First electrode 40 is electrically coupled to drive voltage circuit 144by first electrode lead 146 soldered to first electrode 40 at a firstelectrode soldering area 128. Second electrode 42 is grounded by way ofa first ground lead 148 that is connected to second electrode 42 at asecond electrode soldering area 129.

First and second sensor electrodes, 130 and 132, are electricallycoupled to each other and to drive voltage circuit 144 through afeedback circuit 150. A first sensor electrode lead 152 connects thefirst sensor electrode 130 to feedback circuit 150. A second sensorelectrode lead 154 connects the second sensor electrode 132 to feedbackcircuit 150.

A second ground lead 156 connects feedback circuit 150 to the ground. Acontrol lead 158 connects feedback circuit 150 to drive voltage circuit144.

As shown by way of example, the specific structural details of oneembodiment of a feedback circuit, such as feedback circuit 150 in FIG.9, and a drive voltage circuit, such as drive voltage circuit 144 inFIG. 9, can be appreciated by reference to FIG. 13.

As shown in FIG. 13 for purposes of illustration, receptacle 24 isfilled with a liquid medium in which the input voltage V_(I) suppliedbetween first electrode 40 and grounded second electrode 42 creates anelectric field.

The actual voltage V_(A) created in the liquid medium in receptacle 24between first sensor electrode 130 and second sensor electrode 132 isillustrated as a voltage drop occurring over a variable resistor 159.Resistor 159 represents the resistance to the electric field presentedin the liquid medium in receptacle 24 between the first and secondpredetermined points in array 38. In operation of a sorting apparatussuch as sorting apparatus 110, the composition of the liquid medium willvary from a number of causes. This as a result varies the electricalresistance of the liquid medium.

The actual voltage V_(A) inherently differs from the input voltage V_(I)by the amount of voltage drop occurring in the liquid medium at twolocations. These are between first electrode 40 and first sensorelectrode 130 and between second sensor electrode 132 and secondelectrode 42. The resistance in the liquid medium in receptacle 24between first electrode 40 and first sensor electrode 130 is illustratedas a resistor 160a, while the corresponding resistance between secondsensor electrode 132 and second electrode 42 is illustrated as aresistor 160b.

FIG. 13 illustrates in addition an exemplary arrangement of circuitelements intended to perform the functions of drive voltage circuit 144and feedback circuit 150 illustrated in FIG. 9.

In an aspect of the present invention discussed relative to sortingapparatus 20, a sorting apparatus, such as sorting apparatus 110, isalso provided with electric force means for generating the electricfield in the fluid medium in receptacle 24. In sorting apparatus 20illustrated in FIG. 1, one example of such an electric force means wasillustrated in the form of battery 44.

In FIG. 9, however, an alternative form of such an electric force meansis illustrated in the form of drive voltage circuit 144. Shown in moredetail in FIG. 13, drive voltage circuit 144 comprises an originalvoltage V_(O) which is coupled through an input resister 161 to thenegative terminal of a differential amplifier 162. In this manner, thevoltage appearing on first electrode lead 146 coupled to the outputterminal of differential amplifier 162 has an inverse polarity to inputvoltage V_(O). A biasing resister 163 is coupled in parallel between thenegative input terminal of differential amplifier 162 and the outputterminal thereof.

While in some embodiments, input voltage V_(O) may comprise a battery,it is also the intention in sorting apparatus 110 to afford for an inputvoltage V_(O), which can itself be variable and which, due to thecoupling thereof through the negative input terminal of differentialamplifier 162, is inversely variable relative to the input voltage V_(I)that is eventually supplied over first electrode lead 146 to firstelectrode 40.

According to one aspect of the present invention, a sorting apparatus,such as sorting apparatus 110 illustrated in FIG. 9, includes sensormeans for detecting the intensity of the electric field generated withinthe liquid medium in receptacle 24 in any preselected portion of array38. The electric field detected corresponds to actual voltage V_(A)illustrated in FIG. 13. In FIG. 13 the preselected portion of array 38over which actual voltage V_(A) is measured is located between a firstpredetermined point 134 in array 38 corresponding to first sensorelectrode 130 and a second predetermined point 135 therein correspondingto second sensor electrode 132.

FIG. 13 illustrates an example of circuit elements capable of performingthe function of such a sensor means for use in a sorting apparatusincorporating teachings of the present invention. These elements includefirst sensor electrode 130 positioned within array 38 of obstacles 39 atfirst predetermined point 134 and a second sensor electrode 132positioned within array 38 at second predetermined point 135. Incombination therewith, the sensor apparatus according to the teachingsof the present invention comprises control means coupled to first sensorelectrode 130 and second sensor electrode 132 for maintaining theelectric field in the liquid medium in receptacle 24 at a predeterminedintensity.

The elements of one embodiment of such a control means are shown in FIG.13 in the form of the circuit components and functional groupingsthereof that comprise feedback circuit 150. Feedback circuit 150functions to vary the voltage supplied by drive voltage circuit 144 tofirst electrode 40 utilizing a control signal supplied thereto overcontrol lead 158. While the elements of feedback circuit 150 will bedescribed in detail subsequently, the effect of the control signalsupplied over control lead 158 to drive voltage circuit 144 will bebetter appreciated fully by an initial discussion of the constituentelements of drive voltage circuit 144.

The control signal from control lead 158 is applied to the positiveinput terminal of differential amplifier 162 through a second inputresistor 164. The effect of the control signal on control lead 158 is tovary the output of drive voltage circuit 144 on first electrical lead146 with the object of stabilizing actual voltage V_(A). To do so theintensity of the electric field in the fluid medium in receptacle 24 isincreased, when the control signal indicates that the actual voltageV_(A) is less than some predetermined referenced voltage desired by theoperator of sorting apparatus 110. Correspondingly, the control signalof control lead 158 is oppositely polarized and thus decreases theintensity of the electric field in the liquid medium in receptacle 24,when the control signal reflects that the actual voltage V_(A) isgreater than that same predetermined reference voltage. In this manner,the control signal supplied on control lead 158 to drive voltage circuit144 will by the action of differential amplifier 162 adjust the actualeffect of original voltage V_(O) so as to maintain the actual voltageV_(A) at any desired level.

The use of the control signal supplied over control lead 158 to drivevoltage circuit 144 could be utilized as a mechanism for effectingdesired variations in the voltage supplied to first electrode 40 onfirst electric lead 146. Under most circumstances, however, it isanticipated that the known propensity of a liquid medium in whichmicrostructures are migrating will vary during the time of operation dueto a number of factors, such as evaporation, chemical reactions, andtemperature changes. An initial objective of the circuitry that will nowbe described relative to feedback circuit 150 is to compensate for whatis in effect the changeable nature of the liquid medium in receptacle 24as illustrated by variable resistor 159. In this manner actual voltageV_(A) is maintained at some predetermined constant intensity.

As illustrated in FIG. 13, feedback circuit 150 includes a differentialamplifier circuit 166 having a first input terminal 167, a second inputterminal 168, and an output terminal 169. First input terminal 167 iscoupled through a first buffer amplifier circuit 170 to first sensorelectrode 130, while second input terminal 168 is coupled through asecond buffer amplifier circuit 171 to second sensor electrode 132.

First buffer amplifier circuit 170 is comprised of a differentialamplifier 172 connected in the manner illustrated between the circuitcomponents already described above. Correspondingly, second bufferamplifier circuit 170 is comprised of a differential amplifier 173connected as illustrated. It is the function of first and secondamplifier circuits 170, 171, respectively, to serve as impedance buffersfor first and second input terminals 167, 168, respectively, ofdifferential amplifier circuit 166.

Within differential amplifier circuit 166, first input terminal 167 iscoupled through an input resistor 174 to the negative input terminal ofa differential amplifier 175, while second input terminal 168 is coupledto the positive terminal thereof through an input resistor 176.Resistors 177 and 178 are connected as shown in FIG. 13 to biasdifferential amplifier 175 into the desired operator thereof. By thearrangements illustrated and described, differential amplifier circuit166 produces at output terminal 169 thereof an output signal thatcorresponds to the intensity of actual voltage V_(A) of the electricfield in the liquid medium in receptacle 24.

According to another aspect of the present invention, a feedbackcircuit, such as feedback circuit 150, includes a comparator meanscoupled to output terminal 169 of differential amplifier circuit 166 forproducing a control signal at control lead 158 that reflects thedifference between the output signal on output terminal 169 and areference voltage reflecting a predetermined desired intensity of actualvoltage V_(A).

As shown by way of example and in FIG. 13, such a reference voltage issupplied by a reference voltage circuit 179 which comprises adifferential amplifier 180 having a reference voltage V_(R) coupled tothe positive input terminal thereof through a variable resistor 181. Inthis manner, variable resistor 181 can be used to adjust the effect ofreference voltage V_(R) appearing at the output side of differentialamplifier 180 at an output terminal 182 for reference voltage circuit179.

It is the purpose of comparison circuit 183 illustrated in FIG. 13 toproduce on control lead 158 a control signal reflecting the difference,if any, between the output signal appearing at output terminal 169 ofdifferential amplifier circuit 166 and the portion of reference voltageV_(R) appearing at output terminal 182 of reference voltage circuit 179.Toward that end, comparison circuit 183 comprises a differentialamplifier 184 coupled at the output terminal thereof to control lead158. The positive input terminal of differential amplifier 184 iscoupled through an input resistor 185 to output terminal 169 ofdifferential amplifier circuit 166, while the negative input terminal ofdifferential amplifier 184 is coupled through an input resistor 186 tooutput terminal 182 of reference voltage circuit 179. Variable resistors187, 188 are connected as shown within comparison circuit 183 to effectdesired biasing of differential amplifier 184.

In the circuitry illustrated in FIG. 13, differential amplifiers 162,172, 173, 175, 180, and 184 can, by way of example, comprise operationalamplifiers available from Analog Devices as Product No. AD795N. Suchdevices utilize field effect transistor inputs and have low noisecharacteristics. The values of the resistors illustrated are as follows:

R₁ =10 kΩ

R₂ =10⁶ Ω

For an apparatus, such as sorting apparatus 110, original voltage V_(O)is equal to negative 15 volts, while reference voltage V_(R) is equal topositive 15 volts.

By means of the circuitry illustrated in FIG. 14, any desiredpredetermined actual voltage V_(A) can be maintained between first andsecond sensor electrodes 130, 132, respectively, despite variations overtime in the nature of the liquid medium in receptacle 24.

It must be noted that although an electric field has been described indetail as the means for inducing migration of the microstructures, otherfields such as hydrodynamic, magnetic, and gravity, for example, mayalso be used.

B. Cell Fractionation

FIGS. 14-18 illustrate another use of the teachings of the presentinvention to facilitate the study of the motion of cells, such as humanred blood cells, bacterial cells, and cancer cells, for example, throughchannels in a single layer and in single file. For red blood cells, thechannels may simulate those found in capillaries, the lung alveoli, andthe spleen in the human body. Further, with the apparatus of the presentinvention, red blood cells can be fractionated on the basis of physicalproperties which are otherwise difficult to probe by biological markers.

The apparatus within the scope of the present invention compriseschanneling means positioned within receptacle 24 for allowing passage ofcells through receptacle 24 in essentially a single layer in singlefiles.

One possible configuration of an array for all fractionation within thescope of the present invention is illustrated in FIG. 14. This array 192is called a percolating array and is patterned as a maze. In thisconfiguration, the channeling means comprises obstacles 193 positionedupstanding from floor 28 of receptacle 24 in various connectingpositions to form open areas 194, passageways 196, and dead ends 197,such as are found in mazes. As can be seen in FIG. 14, cells 199 migratethrough percolating array 192 through open areas 194 and passageways 196and are at times blocked by dead ends 197. Passageways 196 may be madelinear, curved, or whatever shape desired so as to be able to observemigration of cells through variously shaped passageways. Passageways 196may have a width in the range of from about 1.0 micron to about 10.0microns and a depth with the range of from about 1.0 micron to about10.0 microns. Cells migrating in single file can be seen labelled as199a.

Percolation, as herein discussed, is the phenomenon facilitated byincreasing path connectedness in an array of obstacles due to randomaddition of discrete segments of allowed motion through the obstacles.At the percolation threshold, there is just one path on the averagethrough the array, with all other paths leading to dead ends. Theability of cells to find that path can be observed with the percolatingarrays 192 of the present invention.

Within the scope of the present invention, percolating arrays 192 havebeen constructed on a rectangular lattice in a preferred percolatingembodiment. A single computer algorithm fills some fraction of thelattice with lines, for example, 40% so as to form the variety of openareas 194 and passageways 196. The computer program is then made intothe opaque mask and the microlithographic process as earlier describedis carried out.

In the preferred embodiment, the obstacles 193 are comprised of barriers5.0 microns long and 1.0 micron wide. The preferred etch depth ofpercolating array 192 is 0.35 microns. FIG. 14 illustrates an enlargedsection of such a photomicrograph percolating array 192.

One example of the use of percolating array 192 is for study of themovements of cells, such as E. Coli, from one end of array 192 to theother. In one experiment, E. Coli cells were placed at the first end 32of receptacle 24 while food was placed at the second end 34 ofreceptacle 24. The E. Coli cells were then observed as they migrated ina single layer through percolating array 192 from first end 32 towardsecond end 34. When dead ends 197 were reached by the E. Coli cells, themanner in which the E. Coli cells reoriented themselves in order to moveaway from the dead ends 197 was observed. Also observed was the abilityof the E. Coli cells to find an open path from the starting point atfirst end 32 to the food at second end 32.

The studies conducted for the E. Coli cells can also be conducted formany other types of cells. Percolating arrays 192 can be used to studythe manner in which many other types of free floating cells reorientthemselves in a fluid suspension when confronted with barriers andpassageways, and the manner in which various passageways are chosen.

As earlier stated, the percolating arrays 192 are formed such thatmigration of cells in a single layer and in single files can beobserved. Therefore, in order to accommodate the various sizes of cellsto be observed, the size of open areas 194 and passageways 196 in eacharray 192 can be designed as needed. The pattern of the array can alsobe designed as desired. Any pattern can be produced and reproduced.

One additional important aspect of percolating arrays 192 is the abilityto perform electrophoresis of charged spherical balls within percolatingarrays 92. The mobilities of even simple balls are rich in a percolatingarray because of the numerous dead-ends that exist in a percolatingarray near the percolating threshold. If the electric fields are toobig, then the balls cannot back-diffuse out of the dead end against theapplied electric force. Hence, mobility shuts down above a criticalfield. Measuring the diffusion of fluorescent balls of precise diameterwill allow study of a diffusion of polymers in arrays.

Referring now to FIG. 15, another embodiment of the present inventioncan be seen. In FIG. 15, an array 200 of obstacles in the form ofelongated rectangular bunkers 202 is positioned within receptacle 24.Bunkers 202 are comprised of a rectangular shape having opposingsidewalls 203 and a top 204. Bunkers 202 upstand from floor 28 ofreceptacle 24. Bunkers 202 are positioned within columns and rows withinreceptacle 24. Cells migrate through the columns and between the rows ofbunkers 202 in a migration direction indicated by arrow M. Thelongitudinal axis of the bunkers are disposed in alignment withmigration direction M. Channels 206 are formed between rows of bunkers202 through which the cells migrate. A separation distance, S_(r),between rows of bunkers 202, indicates the size of channels 206.

While the size and organization of bunkers 202 may vary, in a preferredembodiment within the scope of the present invention, the separationdistance S_(r) is sized to allow the cells to migrate through channels206 in essentially a single layer and in single files.

The height H of each bunker 202 should also be such that it allows thecells to pass through the bunkers 202 in essentially a single layer. Aswith the apparatus for fractionating DNA, a coverslip 36 is fused to thetops 204 of bunkers 202 so as to prevent migration of cells between thecoverslip and the tops 204 of bunkers 202 to ensure that the cellsmigrate through the array 200 of bunkers 202 in essentially a singlelayer.

While bunkers 202 are the preferred obstacles for forming channels 206,different structures may also be used to simulate channels through whichthe cells can migrate and be observed. These alternate structures arealso within the scope of the present invention.

FIG. 16 illustrates an apparatus 212 for cell sorting and fractionation.As an example, and not as a limitation, cells 214 are shown migratingthrough array 200 of bunkers 202. Cells 214 can be seen moving betweenthe rows of bunkers 202 through channels 206. Some cells begin round,deform to fit within channels 206, and then regain their shape once outof the channel. Other cells which may have lost some degree ofdeformability, however, do not regain their shape, or are misshapeninitially. Some are even trapped in these, restricted channels. This, asearlier stated, can be caused by aging, sickling or other in vivo or invitro problems.

For illustration, cells 214 are shown to be disc shaped. As cells 214enter channels 206, cells 214 deform from a disc shape to an elongatedshape so as to be able to squeeze through channels 206. When cells 214are positioned between bunkers 202 and within channels 206, cells 214have a thin elongated shape. As cells 214 move from between bunkers 202and into open space, the healthy cells 214 can be seen to resume theiroriginal disc shapes. The unhealthy cells may be found to not be able toresume their original shapes because of a lack of plastic flow. By theapparatus of the present invention, the flexibility and deformability ofred blood cells can be studied.

FIGS. 17A-17E illustrate an individual cell 214 moving through a pair ofbunkers 202. As shown in FIG. 17A, before passing through bunkers 202,the cell 214 is perfectly disc shaped. In FIG. 17B, cell 214 is seenbeginning to deform in order to fit between bunkers 202 in channel 206.FIG. 17C illustrates cell 214 deformed into an elongated thin shape tofit within channel 206. As shown in FIG. 17D, as cell 214 begins to moveout of channel 206, cell 214 begins to regain its original disc shape.Once completely out of channel 206, as shown in FIG. 17E, the elasticityof cell 214 allows cell 214 to completely regain its original discshape.

In contrast, FIGS. 18A-18B illustrate an unhealthy cell 216 whoseelastic properties have been lost. Although unhealthy cell 216 has anoriginal round disc shape of a healthy cell, its flexibility isdiminished such that it cannot deform to fit into channel 206. As cell216 passes through channel 206, cell 216 cannot deform into a thinelongated shape to fit into channel 206 and becomes stuck in the openingof channel 206. In the case of cancer cells, it is thought that wherethe cancer cells become stuck, a new tumor is grown. The activity ofcancer cells can be studied with the teachings of the present invention.

Thus, it can be seen that by using the apparatus of the presentinvention, the elasticity and flexibility of cells can be studied.Further, the consequences of lack of plastic flow of the cells can beobserved and studied. Further, still, the amount of energy consumed bythe cell to deform and regain its shape can easily be measured andrecorded.

Another important advantage and use of the present invention is to studyand observe the physical properties of cells in a variety of chemicalenvironments. Array 200 can be exposed to various chemical environments,such as irradiation, light illumination, or sickling phenomenaimitations, before allowing the cells to migrate through array 200. Thereactions of cells as they migrate through these various environmentscan then be studied. For example, experiments can be designed todetermine what kinds of chemical reactions cause aging of the cells anddestroy ability of cells to be flexible. Other experiments can bedesigned and conducted to determine the chemical effects on cancercells. Ultimately, an unlimited number of cellular effects can beobserved.

Also advantageous, the experiments can be easily repeated to verify dataor to make minor changes to the experimental controls. Thus, cells canbe sorted by desired physical properties, that is, by their reactions tovarious environments. As the cells are sorted, they can be separated andcollected.

Another important advantage of the present invention with regard tostudying cells is the reproducibility and repeatability of the array ofobstacles. Since the arrays 200 can consist of obstacles which arerepeated thousands of times, even subtle variations in small quantitiesin the membrane of the cell can be amplified. Additionally, by theapparatus of the present invention, many individual cells can beobserved at once as they migrate through the channels 206 of theapparatus. Observation of more than just one cell is possible.

With regard to mobility of the cells through the apparatus of thepresent invention, cells can be migrated through array 200 using variousfields. For example, migration can be caused by flowing fluid throughthe array in a hydrodynamic field through flow cytometry wherein waterpressure is used to force the cells through the array. The cells mayalso be induced to move by a gravity field. Alternatively, magneticbeads may be placed on the apparatus to create a magnetic field toinduce movement of the cells. Further, focused beams of light referredto as optical tweezers may be used to move the cells through array 200.Other means for inducing the cells to migrate through the array 200 arealso within the scope of the present invention.

As one example of an embodiment within the scope of the presentinvention, the apparatus can be designed to simulate capillaries in thehuman body by having channeling means positioned within receptacle 24which mimic the openings that the blood cell must pass through in thebody. By precise control of chemical environment, channel opening andtopology, flow velocity, and the application of theories of membranephysics, understanding can be obtained of how cells pass through complexenvironments, cell aging, and how the chemical environment of the cellsolution controls the membrane properties.

The invention may be embodied in other specific forms without departingfrom its spirit other essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

What is claimed is:
 1. An apparatus for sorting microstructures, in afluid medium, said apparatus comprising:a. a substrate having areceptacle located on a side thereof, said receptacle having first andsecond ends and a floor bound on opposite sides by a pair of upstandingopposed side walls extending between said first and second ends of saidreceptacle, migration of the microstructures from the first end of saidreceptacle to said second end of said receptacle defining a migrationdirection for said receptacle; b. a two-dimensional array of obstaclespositioned in said receptacle intermediate said first and second endsthereof; and c. ceiling means positioned over and contacting said arrayof obstacles for precluding migration of the microstructures betweensaid array of obstacles and said ceiling means.
 2. An apparatus asrecited in claim 1, wherein said obstacles are upstanding from saidfloor of said receptacle in a plurality of rows traversing saidmigration direction.
 3. A sorting apparatus as recited in claim 2,wherein each of said obstacles has a top at the end thereof oppositefrom said floor of said housing, and said top of each of said obstaclesis permanently secured to said ceiling means.
 4. A sorting apparatus asrecited in claim 2, wherein said obstacles of said array are arranged ina preselected and reproducible pattern, each obstacle in said array ofobstacles having a height measured in a direction normal to said floorof said receptacle, a length measured in a direction parallel to saidmigration direction, and a width measured in a direction normal to saidmigration direction, and wherein the distance of each of said obstaclesfrom an adjacent of said obstacles defines a separation distancetherebetween.
 5. A sorting apparatus as recited in claim 4, wherein saidheight of each of said obstacles is in a range of from about 0.01microns to about 20.0 microns.
 6. A sorting apparatus as recited inclaim 4, wherein said height of each of said obstacles is in a range offrom about 0.01 microns to about 0.50 microns.
 7. A sorting apparatus asrecited in claim 4, wherein said height of each of said obstacles is ina range of from about 1.0 micron to about 5.0 microns.
 8. A sortingapparatus as recited in claim 4, wherein said separation distance issubstantially equal to a radius of gyration of each of themicrostructures.
 9. A sorting apparatus as recited in claim 4, whereinsaid separation distance is in a range of from about 0.01 microns toabout 50.0 microns.
 10. A sorting apparatus as recited in claim 4,wherein said separation distance is in a range of from about 0.01microns to about 1.0 micron.
 11. A sorting apparatus as recited in claim4, wherein said separation distance is in a range of from about 1.0micron to about 20.0 microns.
 12. A sorting apparatus as recited inclaim 4, wherein said separation distance is substantially equal to saidlength of said obstacles.
 13. A sorting apparatus as recited in claim 4,wherein said obstacles comprise posts.
 14. A sorting apparatus asdefined in claim 4, wherein said obstacles comprise elongated bunkers.15. A sorting apparatus as recited in claim 14, wherein the longitudinalaxis of said bunkers are disposed in alignment with said migrationdirection of said receptacle.
 16. A sorting apparatus as defined inclaim 4, wherein said obstacles comprise elongated interconnectedbunkers forming an open lattice of nonlinear channels between said firstand said second end of said receptacle.
 17. A sorting apparatus asrecited in claim 2, wherein the top of each of said obstacles is ionfusion bonded to said ceiling means.
 18. An apparatus as recited inclaim 2, wherein said obstacles in adjacent of said rows thereof arealigned along said migration direction.
 19. An apparatus as recited inclaim 2, wherein said obstacles in adjacent of said rows thereof areoffset relative to said migration direction.
 20. An apparatus as recitedin claim 2, wherein said rows of said obstacles are parallel to eachother.
 21. An apparatus as recited in claim 2, wherein said rows of saidobstacles are perpendicular to said migration direction.
 22. Anapparatus as recited in claim 2, wherein said rows of said obstacles areoriented at an angle to said migration direction.
 23. An apparatus asrecited in claim 22, wherein said angle has a measure of about 45°. 24.An apparatus as recited in claim 2, wherein spaces between adjacent ofsaid obstacles in a cross section of said array of said obstacles takennormal to said floor of said receptacle define pores of a latticestructure, and said pores of said lattice structure are uniform in sizeand shape throughout said array of said obstacles.
 25. An apparatus asrecited in claim 2, wherein spaces between adjacent of said obstacles ina cross section of said array make a normal to said floor of saidreceptacle define pores of a lattice structure, and said pores of saidlattice structure vary in size and shape throughout said array.
 26. Asorting apparatus as defined in claim 1, wherein said obstaclescumulatively comprise a percolating array.
 27. A sorting apparatus asrecited in claim 1, wherein said ceiling means comprises a coverslipextending across said receptacle from one of said pair of upstandingopposing side walls to the other.
 28. A sorting apparatus as recited inclaim 27, wherein said coverslip is transparent.
 29. A sorting apparatusas recited in claim 1, wherein the height of said side walls of saidreceptacle as measured normal to said floor of said receptacle definethe depth of said receptacle, and said receptacle is so configured thatsaid depth thereof causes the microstructures to migrate through saidreceptacle in said migration direction in essentially a single layer.30. An apparatus as recited in claim 1, wherein migration of themicrostructures through said obstacles is in said migration direction.31. An apparatus as recited in claim 1, wherein said obstacles areupstanding from said floor of said receptacle in a uniform pattern. 32.An apparatus as recited in claim 1, wherein said obstacles areupstanding from said floor of said receptacle in a staggered pattern.33. A sorting apparatus for fractionating microstructures, in a fluidmedium, said sorting apparatus comprising:a. a substrate having areceptacle located on a side thereof, said receptacle having first andsecond ends and a floor bound on opposite sides by a pair of upstandingopposed side walls extending between said first and second ends of saidreceptacle, migration of the microstructures from the first end of saidreceptacle to said second end of said receptacle defining a migrationdirection for said receptacle; b. sifting means positioned within saidreceptacle intermediate said first and second ends thereof traversingsaid migration direction for interacting with the microstructures topartially hinder migration of the microstructures in said migrationdirection in the fluid medium, said sifting means comprising an array ofobstacles upstanding from said floor of said receptacle, and arranged ina preselected and reproducible pattern, each of said obstacleshaving:(i) a v-shaped cross section in a plane disposed parallel to saidfloor of said receptacle, said v-shaped cross section of said obstaclescomprising first and second arms intersecting at an end of each to forma vertex of said v-shaped cross section, the other ends of said firstand second arms remote from said vertex forming an open end of saidv-shaped cross section; (ii) a top at the end thereof opposite from saidfloor of said receptacle; (iii) a height measured in a direction normalto said floor of said receptacle; (iv) a length measured in a directionparallel to said migration direction; and (v) a width measured in adirection normal to said migration direction, (vi) the distance of eachof said obstacles from an adjacent of said obstacles defining aseparation distance therebetween; and c. ceiling means positioned oversaid sifting means for covering said receptacle and for causingmigration of the microstructures in said receptacle to occur throughsaid sifting means exclusively.
 34. A sorting apparatus as recited inclaim 33, wherein the open end of the v-shaped cross section of saidobstacles is disposed opposing said migration direction of saidreceptacle.
 35. A sorting apparatus for fractionating microstructures ina fluid medium, said sorting apparatus comprising:a. a substrate havinga receptacle located on a side thereof, said receptacle having first andsecond ends and a floor bound on opposite sides by a pair of upstandingopposed side walls extending between said first and second ends of saidreceptacle, migration of the microstructures from the first end of saidreceptacle to said second end of said receptacle defining a migrationdirection for said receptacle; b. sifting means positioned within saidreceptacle intermediate said first and second ends thereof traversingsaid migration direction for interacting with the microstructures topartially hinder migration of the microstructures in said migrationdirection in the fluid medium, said sifting means comprising an array ofobstacles upstanding from said floor of said receptacle and arranged ina preselected and reproducible pattern, each of said obstacleshaving:(i) a cup-shaped cross section in a plane disposed parallel tosaid floor of said receptacle, said cup-shaped cross section of saidobstacles comprising:(A) a first leg and a second leg positioned inopposed relationship substantially parallel to said migration direction,each of said first and second legs having thereby oppositely disposedfirst and second ends; and (B) a third leg positioned substantiallyperpendicular to said migration direction and being connected betweensaid first ends of each of said first and second legs, thereby to definebetween said second ends of said first and second legs an open end ofsaid cup-shaped cross section; (ii) a top at the end thereof oppositefrom said floor of said receptacle; (iii) a height measured in adirection normal to said floor of said receptacle; (iv) a lengthmeasured in a direction parallel to said migration direction; and (v) awidth measured in a direction normal to said migration direction, (vi)the distance of each of said obstacles from an adjacent of saidobstacles defining a separation distance therebetween; and c. ceilingmeans positioned over said sifting means for covering said receptacleand for causing migration of the microstructures in said receptacle tooccur through said sifting means exclusively.
 36. A sorting apparatus asrecited in claim 35, wherein said open end of said cup-shaped crosssection of said obstacles is disposed opposing said migration directionof said receptacle.
 37. An apparatus for sorting microstructures in afluid medium, said apparatus comprising:a. a substrate having areceptacle located on a side thereof, said receptacle having first andsecond ends and a floor bound on opposite sides by a pair of upstandingopposed side walls extending between said first and second ends of saidreceptacle, the height of said side walls defining a depth of saidreceptacle; b. a two-dimensional array of obstacles upstanding from saidfloor of said receptacle, each of said obstacles in said array having aheight measured in a direction normal to said floor of said receptacle,said height of said obstacles being substantially equal to said depth ofsaid receptacle; and c. ceiling means positioned over said array ofobstacles for covering said receptacle and for causing the migration ofthe microstructures within said receptacle to occur exclusively throughsaid array of obstacles, the end of each of said obstacles remote fromsaid floor of said receptacle engaging said ceiling means, thereby topreclude migration of the microstructures between said ceiling means andeach of said obstacles.
 38. A sorting apparatus as recited in claim 37,wherein said ceiling means comprises a coverslip extending across saidreceptacle from one of said pair of upstanding opposing side walls tothe other.
 39. A sorting apparatus as recited in claim 38, wherein saidcoverslip and said substrate are comprised of materials havingsubstantially similar coefficients of thermal expansion.
 40. Anapparatus as recited in claim 38, wherein said coverslip is comprised ofa ceramic.
 41. An apparatus as recited in claim 38, wherein saidcoverslip is comprised of quartz.
 42. An apparatus as recited in claim38, wherein said coverslip is comprised of sapphire.
 43. An apparatus asrecited in claim 38, wherein said coverslip is comprised of pyrex. 44.An apparatus as recited in claim 38, wherein said coverslip is comprisedof silicon.
 45. A sorting apparatus as recited in claim 38, wherein saidcoverslip is transparent.
 46. A sorting apparatus as recited in claim38, wherein said coverslip is ion fusion bonded to said pair ofupstanding opposing side walls.
 47. An apparatus as recited in claim 37,wherein said substrate is comprised of a material that can be subjectedto photolithographic etching.
 48. An apparatus as recited in claim 37,wherein said substrate is comprised of silicon.
 49. An apparatus asrecited in claim 37, wherein said substrate is comprised of quartz. 50.An apparatus as recited in claim 37, wherein said substrate is comprisedof sapphire.
 51. A sorting apparatus as recited in claim 37, whereinsaid depth of said receptacle and said height of said obstacles is suchthat when the microstructures are caused to migrate in the fluid mediumfrom said first end of said receptacle to said second end of saidreceptacle, the microstructures do so in essentially a single layer. 52.A sorting apparatus as recited in claim 37, wherein said height of eachof said obstacles is in a range of from about 0.01 microns to about 20.0microns.
 53. A sorting apparatus as recited in claim 37, wherein saidheight of said obstacles is in a range of from about 0.01 microns toabout 0.50 microns.
 54. A sorting apparatus as recited in claim 37,wherein said height of said obstacles is in a range of from about 1.0micron to about 5.0 microns.
 55. A lattice structure of predeterminedpore size and shape for use in sorting individual microstructures in afluid medium, said lattice structure comprising:a. a substrate havingthereon a planar surface, said planar surface defining a floor of saidlattice structure; b. a coverslip disposed parallel to and separatedfrom said floor, thereby to define between said coverslip and said floora migration channel capable of receiving the microstructures in thefluid medium for migration of the microstructures through said migrationchannel; c. a two-dimensional array of obstacles extending between andcontacting said floor and said coverslip in a preselected andreproducible pattern, the space between adjacent of said obstacles in across section of said array taken normal to said floor of said latticestructure defining pores of said lattice structure, said pores of saidlattice structure being so sized and configured as to slow the rate ofmigration of the microstructures.
 56. A lattice structure as recited inclaim 55, wherein said obstacles are integrally formed with saidsubstrate.
 57. A lattice structure as recited in claim 55, wherein saidsubstrate, said coverslip, and said obstacles are comprised of materialshaving substantially similar coefficients of thermal expansion.
 58. Alattice structure as recited in claim 56, wherein said obstacles areintegrally formed at one end thereof with said substrate and bonded atthe other end thereof to said coverslip.
 59. A lattice structure asrecited in claim 55, wherein said pores of said lattice structure assumea generally rectangular shape.
 60. A lattice structure as recited inclaim 55, wherein said coverslip is transparent.
 61. A lattice structureas recited in claim 55, wherein said coverslip affords visualobservation of the migration of the microstructures.
 62. A latticestructure as recited in claim 55, wherein each of said obstacles in saidarray has a top at the end thereof opposite from said floor of saidlattice structure, and said coverslip is ion fusion bonded to said topof each of said obstacles in said array.
 63. A lattice structure asrecited in claim 55, wherein the depth of said migration channel isdefined by the distance between said floor and said cover slip measurednormal to said floor, and said depth of said migration channel is suchas to cause migration of the microstructures in said migration channelto occur in essentially a single layer.
 64. A lattice structure asrecited in claim 55, wherein said pores of said lattice structure are soconfigured as to cause the microstructures to migrate through saidlattice structure in essentially a single file.
 65. A lattice structureas recited in claim 64, wherein said pores of said lattice structure areso configured as to cause the microstructures to migrate through saidlattice structure in essentially a single file.
 66. A lattice structureas recited in claim 55, wherein each obstacle in said array of obstacleshas a height measured in a direction normal to said floor of saidmigration channel, and said height of each of said obstacles is in arange of from about 0.01 microns to about 20.0 microns.
 67. A latticestructure as recited in claim 55, wherein each obstacle in said array ofobstacles has a height measured in a direction normal to said floor ofsaid migration channel, and said height of each of said obstacles is ina range of from about 0.01 microns to about 0.50 microns.
 68. A latticestructure as recited in claim 55, wherein each obstacle in said array ofobstacles has a height measured in a direction normal to said floor ofsaid migration channel, and said height of each of said obstacles is ina range of from about 1.0 micron to about 5.0 microns.
 69. A latticestructure as recited in claim 55, wherein said array of obstaclescomprise a plurality of rows of said obstacles traversing said migrationchannel.
 70. A lattice structure as recited in claim 69, wherein saidrows of obstacles are parallel to each other.
 71. A lattice structure asrecited in claim 55, wherein said obstacles are disposed in a uniformpattern.
 72. A lattice structure as recited in claim 55, wherein saidobstacles is disposed in a staggered pattern.
 73. A lattice structure asrecited in claim 55, wherein said pores of said lattice structure areuniform in size and shape throughout said array of said obstacles.
 74. Alattice structure as recited in claim 55, wherein said migration channelhas a depth measured between said floor of said lattice structure andsaid coverslip in a range from about 0.01 microns to less than about 0.1microns.
 75. A lattice structure as recited in claim 55, wherein saidmigration channel has a depth measured between said floor of saidlattice structure and said coverslip in a range from about 0.01 micronsto about 0.06 microns.
 76. A reusable apparatus for sorting individualmicrostructures in a fluid medium, said apparatus comprising:a. asubstrate having a receptacle located on a side thereof, said receptaclehaving first and second ends and a floor bound on opposite sides by apair of upstanding opposed side walls extending between said first andsecond ends of said receptacle, the height of said side walls defining adepth of said receptacle, said depth of said receptacle beingcommensurate with the size of the microstructures in the fluid medium,whereby when the microstructures are caused to migrate in the fluidmedium from said first end through said second end of said receptaclethe microstructures do so in essentially a single layer; and b. atwo-dimensional array of obstacles positioned in said receptacleintermediate said first and second ends thereof, said obstacles beingcomprised of a rigid self-sustaining material that is chemically inertto the microstructures and to the fluid medium in a normal range oftemperatures suitable for fractionating the microstructures; and c.capping means positioned over and contacting said array of obstacles foraffording visual observation of migration of the microstructures in saidmigration direction through said receptacle, and for precludingmigration of the microstructures between said array of obstacles andsaid capping means.
 77. A sorting apparatus as recited in claim 76,wherein said substrate and said array of obstacles are integrallyformed.
 78. A sorting apparatus as recited in claim 76, wherein saidsubstrate and said array of obstacles are comprised of a material thatcan be subjected to photolithographic etching.
 79. A sorting apparatusas recited in claim 76, wherein said substrate and said array ofobstacles are comprised of silicon.
 80. A sorting apparatus as recitedin claim 76, wherein each of said obstacles has a height measured normalto said floor of said receptacle, and said height is substantially equalto said depth of said receptacle.
 81. A sorting apparatus as recited inclaim 76, wherein migration of the microstructures through said hinderedenvironment means is in said migration direction.
 82. An apparatus forchannelling cells through passageways and simultaneously viewing thecells in a fluid medium, said apparatus comprising:a. a substrate havinga receptacle located on a side thereof, said receptacle having a floorbound by a pair of upstanding opposing side walls and a first end and asecond end, the height of said side walls defining a depth of saidreceptacle, said depth of said receptacle being commensurate with thesize of the cells in the fluid medium, whereby when the cells are causedto migrate in the fluid medium through said receptacle, the cells do soin essentially a single layer, the migration of cells from the first endto the second end of said receptacle defining a migration direction forsaid receptacle; b. a two-dimensional array of obstacles positioned insaid receptacle intermediate said first and second ends thereof, saidarray of obstacles allowing passage of the cells through said receptaclein at least one single file; and c. capping means for covering saidreceptacle intermediate said first and second ends thereof and foraffording visual observation of the migration of the cells through saidarray of obstacles and through said receptacle.
 83. An apparatus asrecited in claim 82, wherein passageways are defined between adjacentones of said obstacles.
 84. An apparatus as recited in claim 83, whereinsaid passageways are linear.
 85. An apparatus as recited in claim 83,wherein said passageways are in alignment with said migration direction.86. An apparatus as recited in claim 83, wherein each of saidpassageways has a width in the range of from about 1.0 micron to about10.0 microns.
 87. An apparatus as recited in claim 83, wherein each ofsaid passageways has a depth in the range of from about 1.0 micron toabout 10.0 microns.
 88. An apparatus for channelling cells throughpassageways and simultaneously viewing the cells in a fluid medium, saidapparatus comprising:a. a substrate having a receptacle located on aside thereof, said receptacle having a floor bound by a pair ofupstanding opposing side walls and a first end and a second end, theheight of said side walls defining a depth of said receptacle, saiddepth of said receptacle being commensurate with the size of the cellsin the fluid medium, whereby when the cells are caused to migrate in thefluid medium through said receptacle, the cells do so in essentially asingle layer, the migration of cells from the first end to the secondend of said receptacle defining a migration direction for saidreceptacle; b. channeling means for allowing passage of cells throughsaid receptacle in at least one single file; c. capping means forcovering said receptacle intermediate said first and second ends thereofand for affording visual observation of the migration of the cellsthrough said channeling means and through said receptacle; and d. meansfor measuring the amount of energy consumed during passage of the cellsthrough said channeling means.
 89. An apparatus for channelling cellsthrough passageways and simultaneously viewing the cells in a fluidmedium, said apparatus comprising:a. a substrate having a receptaclelocated on a side thereof, said receptacle having a floor bound by apair of upstanding opposing side walls and a first end and a second end,the height of said side walls defining a depth of said receptacle, saiddepth of said receptacle being commensurate with the size of the cellsin the fluid medium, whereby when the cells are caused to migrate in thefluid medium through said receptacle, the cells do so in essentially asingle layer, the migration of cells from the first end to the secondend of said receptacle defining a migration direction for saidreceptacle; b. channeling means for allowing passage of cells throughsaid receptacle in at least one single file, said channeling meanscomprising passageways that are curved and are positioned within saidreceptacle; and c. capping means for covering said receptacleintermediate said first and second ends thereof and for affording visualobservation of the migration of the cells through said channeling meansand through said receptacle.
 90. A method for manufacturing a sortingapparatus for fractionating and simultaneously viewing individualmicrostructures in a fluid medium, said method comprising the stepsof:a. forming on one side of a substrate a receptacle having a floorbound by a pair of upstanding opposing side walls, the height of saidside walls defining a depth of said receptacle, said depth of saidreceptacle being commensurate with the size of the microstructures inthe fluid medium, whereby when the microstructures are caused to migratein the fluid medium through said receptacle the microstructures do so inessentially a single layer; b. building a two-dimensional array ofupstanding obstacles in a preselected and reproducible pattern on saidfloor of said receptacle, the end of each of said obstacles remote fromsaid floor defining the top of said obstacle; and c. engaging atransparent coverslip to said tops of said obstacles, thereby topreclude migration of the microstructures between said coverslip andsaid tops of obstacles.
 91. A method as recited in claim 90, whereinsaid steps of forming and building are performed simultaneously.
 92. Amethod as recited in claim 90, wherein said step of building comprisesthe steps of:a. positioning a photoresist layer on the surface of saidsubstrate over areas of said substrate corresponding to said tops ofsaid obstacles of said arrays; b. etching said substrate to apreselected depth equal to said depth of said receptacle; and c.dissolving said photoresist layer from said substrate.
 93. A method asdefined in claim 92, wherein said step of etching comprises the step ofion milling said surface of said substrate with said photoresist layerthereon.
 94. A method as defined in claim 92, wherein said step ofetching comprises the step of chemical etching said surface of saidsubstrate with said photoresist layer thereon.
 95. A method as recitedin claim 90, wherein said step of securing comprises the steps of:a.positioning said coverslip over said array of obstacles such that saidcoverslip is in contact with said tops of said obstacles; and b.applying an electric field between said coverslip and said array ofobstacles, thereby causing ions to migrate therebetween.
 96. A method asrecited in claim 95, wherein the applied voltage in said step ofapplying is in a range of from 200 V to 2000 V.
 97. A method as recitedin claim 95, wherein the applied voltage is said step of applying is 1kilovolt.
 98. A method as recited in claim 95, wherein the step ofapplying is conducted in a temperature in a range of from 300° C. to600° C.
 99. A method for sorting microstructures comprising the stepsof:a. placing the microstructures into a fluid medium; b. introducingthe fluid medium with the microstructures therein into a receptaclehaving a first end and a second end and a floor bound by a pair ofupstanding opposing side walls, the height of said side walls defining adepth of said receptacle; c. positioning within said receptacle atwo-dimensional array of obstacles disposed in a preselected andreproducible pattern; and d. inducing the microstructures in the fluidto migrate through said array of obstacles positioned within saidreceptacle.
 100. A method as recited in claim 99, wherein said step ofinducing comprises the step of imposing a hydrodynamic field on saidfluid medium.
 101. A method as recited in claim 99, wherein said step ofinducing comprises the step of imposing a gravity field on themicrostructures.
 102. A method as recited in claim 99, furthercomprising the step of visually observing migration of themicrostructures through said array of obstacles.
 103. A method asrecited in claim 99, wherein said depth of said receptacle is such thatwhen the microstructures migrate in the fluid medium through saidreceptacle, the microstructures do so in essentially a single layer.104. A method as recited in claim 99, wherein said step of inducingcomprises the step of imposing an electric field through said fluidmedium on the microstructures.
 105. A method for sortingmicrostructures, said method comprising the steps of;a. placing themicrostructures into a fluid medium; b. introducing the fluid mediumwith the microstructures therein into a receptacle having a first endand a second end and a floor bound by a pair of upstanding opposing sidewalls, the height of said side walls defining a depth of saidreceptacle; c. positioning within said receptacle an array of upstandingobstacles in a preselected and reproducible pattern; and d. inducing themicrostructures in the fluid to migrate through said array of obstaclespositioned within said receptacle, said step of inducing comprising thesteps of:(i) positioning a first electrode on said floor of saidreceptacle at said first end of said receptacle; (ii) positioning asecond electrode on said floor of said receptacle at said second end ofsaid receptacle; and (iii) coupling a power source between said firstand second electrodes.
 106. A method as recited in claim 105, furthercomprising the steps of:a. positioning a pair of sensing electrodes atfirst and second preselected points in said array of obstacles; b.amplifying the signal obtained on each of said sensing electrodes; andc. differentiating the signals obtained in said step of amplifying toproduce an output signal corresponding to the intensity of said electricfield in said array between said first and second predetermined points.107. A method for sorting microstructures, said method comprising thesteps of:a. placing the microstructures into a fluid medium; b.introducing the fluid medium with the microstructures therein into areceptacle having a first end and a second end and a floor bound by apair of upstanding opposing side walls, the height of said side wallsdefining a depth of said receptacle; c. positioning within saidreceptacle an array of upstanding obstacles in a preselected andreproducible pattern; and d. inducing the microstructures in the fluidto migrate through said array of obstacles positioned within saidreceptacle, said step of inducing comprising the step of focusing lightonto the microstructures.
 108. A method for sorting microstructures,said method comprising the steps of:a. placing the microstructures intoa fluid medium; b. introducing the fluid medium with the microstructurestherein into a receptacle having a first end and a second end and afloor bound by a pair of upstanding opposing side walls, the height ofsaid side walls defining a depth of said receptacle; c. positioningwithin said receptacle an array of upstanding obstacles in a preselectedand reproducible pattern; and d. inducing the microstructures in thefluid to migrate through said array of obstacles positioned within saidreceptacle, said step of inducing comprising the step of imposing amagnetic field on the microstructures.
 109. A sorting apparatus forfractionating microstructures in a fluid medium, said sorting apparatuscomprising:a. a substrate with a receptacle formed in a side thereof,said receptacle having a first end, a second end, and a floor bound onopposite sides by a pair of side walls that extend between said firstend and second end of said receptacle, migration of the microstructuresfrom said first end of said receptacle to said second end of saidreceptacle defining a migration direction for said receptacle; b. atwo-dimensional array of obstacles upstanding from said floor of saidreceptacle in a preselected and reproducible pattern, the spaces betweenadjacent of said obstacles in a cross section of said array taken normalto said floor of said receptacle defining pores of a lattice structurecapable of interacting with the microstructures to differentially slowthe rate of migration of individual types of the microstructures duringmigration of the microstructures in said migration direction throughsaid array of obstacles, each of said pores of said lattice structurehaving dimensions comprising:i. a height of said pores measured in adirection normal to said floor of said receptacle, said height of saidpores of said lattice structure being in a range of from about 0.01microns to about 20.0 microns; and ii. a separation distance of saidpores measured parallel to said floor of said receptacle betweenadjacent of said obstacles, said separation distance of said pores ofsaid lattice structure being in a range of from about 0.01 microns toabout 50.0 microns; and c. a coverslip extending across said receptaclefrom one of said side walls to the other and contacting the end of eachof said obstacles opposite from said floor of said receptacle.
 110. Asorting apparatus as recited in claim 109, wherein said separationdistance of said pores of said lattice structure is in a range of fromabout 0.01 microns to about 1.0 micron.
 111. A sorting apparatus asrecited in claim 109, wherein said separation distance of said pores ofsaid lattice structure is in a range of from about 1.0 micron to about20.0 microns.
 112. A sorting apparatus as recited in claim 109, whereinsaid separation distance of said pores of said lattice structure is in arange of from about 1.0 micron to about 10.0 microns.
 113. A sortingapparatus as recited in claim 109, wherein said height of said pores ofsaid lattice structure is in a range of from about 1.0 micron to about10.0 microns.
 114. A sorting apparatus as recited in claim 109, whereinsaid height of said pores of said lattice structure is in a range offrom about 0.01 micron to about 0.50 microns.
 115. A sorting apparatusas recited in claim 109, wherein said height of said pores of saidlattice structure is in a range of from about 1.0 micron to about 5.0microns.
 116. A sorting apparatus as recited in claim 109, wherein saidheight of said pores of said lattice structure is in a range from about0.01 microns to less than about 0.1 microns.
 117. A sorting apparatus asrecited in claim 109, wherein said height of said pores of said latticestructure is in a range from about 0.01 microns to about 0.06 microns.118. A sorting apparatus for fractionating microstructures in a fluidmedium, said sorting apparatus comprising:a. a substrate having areceptacle located on a side thereof, said receptacle having first andsecond ends and a floor bound on opposite sides by a pair of upstandingopposed side walls extending between said first and second ends of saidreceptacle, migration of the microstructures from the first end of saidreceptacle to said second end of said receptacle defining a migrationdirection for said receptacle; and b. sifting means positioned withinsaid receptacle intermediate said first and second ends thereoftraversing said migration direction for interacting with themicrostructures to partially hinder migration of the microstructures insaid migration direction in the fluid medium, said sifting meanscomprising an array of obstacles upstanding from said floor of saidreceptacle and arranged in a preselected and reproducible pattern, eachof said obstacles having a v-shaped cross section in a plane disposedparallel to said floor of said receptacle, said v-shaped cross sectionof said obstacles comprising:(i) a first arm and (ii) a second armdisposed at an angel to said first arm and intersecting said first armat the vertex of said v-shaped cross section, the ends of said first andsecond arms remote from said vertex forming the open end of saidv-shaped cross section.
 119. A sorting apparatus as recited in claim118, wherein said open end of said v-shaped cross section of saidobstacles is disposed opposing said migration direction of saidreceptacle.
 120. A sorting apparatus for fractionating microstructuresin a fluid medium, said sorting apparatus comprising:a. a substratehaving a receptacle located on a side thereof, said receptacle havingfirst and second ends and a floor bound on opposite sides by a pair ofupstanding opposed side walls extending between said first and secondends of said receptacle, migration of the microstructures from the firstend of said receptacle to said second end of said receptacle defining amigration direction for said receptacle; and b. sifting means positionedwithin said receptacle intermediate said first and second ends thereoftraversing said migration direction for interacting with themicrostructures to partially hinder migration of the microstructures insaid migration direction in the fluid medium, said sifting meanscomprising an array of obstacles upstanding from said floor of saidreceptacle and arranged in a preselected and reproducible pattern, eachof said obstacles having a cup-shaped cross section in a plane disposedparallel to said floor of said receptacle, said cup-shaped cross sectionof said obstacles comprising:(i) a first leg and a second leg positionedin opposed relationship substantially parallel to said migrationdirection, each of said first and second legs having thereby oppositelydisposed first and second ends; and (ii) a third leg positionedsubstantially perpendicular to said migration direction and beingconnected between said first ends of each of said first and second legs,thereby to define between said second ends of said first and second legsthe open end of said cup-shaped cross section.
 121. A sorting apparatusas recited in claim 120, wherein said open end of said cup-shaped crosssection of said obstacles is disposed opposing said migration directionof said receptacle.